CONCLUSIONS/SIGNIFICANCE:

Antenatal exposure to IAI results in precocious switch-on of Hp&HpRP expression. As EONS biomarker, cord blood Hp&HpRP has potential to improve the selection of newborns for prompt and targeted treatment at birth.

Cord blood haptoglobin (Hp) and haptoglobin-related protein (HpRp) immuno-reactivity revealed by Western blot (A&C) and ELISA (B&D) using antibodies reactive with both Hp and HpRP.

(A) Western blot of umbilical vein (UV) serum from 4 preterm newborns of similar gestational age at birth. The newborns in Lanes 1-3 were categorized as having early-onset sepsis (EONS) based on clinical manifestations and hematological indices and all received i.v. antibiotics. The newborn in Lane 4 had a negative sepsis work-up. Blood cultures remained negative for all 4 newborns. The presence of a conspicuous immunoreactive band corresponding to the β-chain (∼42 kDa) in Lanes 1-3 is consistent with our defined switched-on Hp pattern. The absence of this band indicates for a switched-off pattern in Lane 4. The band ∼9 kDa (Lanes 1&2) corresponds to the α1-chain whereas the band at ∼20 kDa (Lanes 2-3) corresponds to the α2-chain. Thus, the cord blood Hp patterns and phenotypes depicted in this gel are: switched-on pattern and Hp1-1 (Lane 1), switched-on pattern and Hp1-2 (Lane 2), switched-on pattern and Hp2-2 (Lane 3) and switched-off pattern and Hp0-0 (Lane 4). In our cohort, among preterm newborns with switched-on Hp pattern (present β-chain, n = 81), the distribution of Hp phenotypes was as follows: 6.0% Hp0-0 (5/81, no α-chain detected), 19.3% (16/81) Hp1-1, 32.5% (26/81) Hp1-2, and 42.1% (34/81) Hp2-2. The higher band intensity of the α2-chain compared to that of the α1-chain suggest that phenotype impacts on total Hp immunoreactivity. (B) Impact of Hp phenotypes on Hp&HpRP immunoreactivity as measured by ELISA. The red line indicates the group's median. Groups assigned different letters are statistically different at a P<0.05 (Kruskal-Wallis ANOVA). (C) Western blot of 3 representative maternal (Mat) and UV serum retrieved from women with normal deliveries at term. Note the switched-off pattern of the cord blood in contrast to the switched-on pattern of the adult blood. The Hp phenotypes of the mothers are Hp2-2 (Cases #1 and #2) and Hp1-2 in Case #3. (D) Quantitative comparison of Hp&HpRP immunoreactivity measured by ELISA in 19 UV and maternal blood (MB) serum from 19 normal deliveries at term relative to Hp&HpRP immunoreactivity measured in preterm UV with either switched-off or switched-on Hp pattern. The red line indicates the group's median. Groups assigned different letters are statistically different at a P<0.05 (Kruskal-Wallis ANOVA).

Cases (n = 180) were stratified by clinical diagnosis of early-onset neonatal sepsis (EONS, Yes or No) and by haptoglobin (Hp) switch pattern (ON or OFF). The red line indicates the group's median. Groups sharing at least one common letter are statistically not different at a P>0.05 (Kruskal-Wallis ANOVA).

Results of Latent Cluster Analysis (LCA) applied to the population of preterm newborns in the study.

(A) Profile plot of the 2-cluster model solution segregating the preterm newborn in this study (n = 180) by probability of antenatal exposure to intra-amniotic infection and/or inflammation (IAI). The x-axis lists the discriminative indicators and significant covariates with their modal characteristic (in red) for which the probability level expected to manifest in each of the two latent clusters is displayed on the y-axis. Newborns in cluster-1 are characterized by low probability of a switch-on haptoglobin (Hp) pattern, IL-6≥100 pg/mL hematological indices consistent with presumed early-onset sepsis (EONS). In contrast, newborns in cluster-2 are characterized by high probability of Hp switch-on pattern along with increased probabilities for cord blood IL-6≥100 pg/mL and for presumed EONS.

Clustering flowchart based on probability of “antenatal IAI exposure”.

Posterior probabilities for inclusion in cluster-2 were calculated for all 8 possible combinations of indicators (haptoglobin & haptoglobin [Hp&HpRP] switch pattern, cord blood IL-6, presumed EONS) and their modal characteristics. The number of newborns presenting each combination is included in parentheses. All newborns with cluster-2 probability levels >50% (black boxes) were considered “likely exposed”. Alternatively, a diagnosis of “likely non-exposed” was assigned to newborns with cluster-2 probability levels <50% (grey boxes).

Results of 2nd-level validation against indicators of adverse neonatal outcome and impact of expressed Hp phenotype.

(A) Forest plot illustrating the impact of newborn classification on prediction of neonatal complications (2nd-level validation). Open circles indicate the log odds ratios based on each newborn's clinical diagnosis of EONS. Closed squares indicate the log odds ratios based on assignment of newborns to either cluster-1 or cluster-2 based on a probability level of >50%. The length of each line shows the 95% confidence interval. The number of newborns affected by each adverse outcome and levels of statistical significance are included in Table 4. The asterisks (*) indicate major adverse outcomes taken into account in the composite variable.